The goals of this project are to define the mechanisms by which oxidative DNA damage regulates telomeric DNA structure, access to telomeric proteins, telomerase activity and telomere length homeostasis. Dysfunctional redox regulation is common among cancers and elevates reactive oxygen species, which generate mutagenic and cytotoxic DNA lesions. Telomere maintenance is essential for genome stability, yet they are highly susceptible to oxidative damage. Most cancers prevent telomere erosion by upregulating telomerase. This enzyme extends the telomeric single-stranded overhang, which can self-fold into stable secondary structures. We will test the hypothesis that oxidative DNA lesions increase the dynamic flexibility in the telomeric overhang thereby altering accessibility and telomerase activity. For this, we developed a single molecule fluorescence resonance energy transfer detection system that measures structural dynamics in overhang conformation and telomerase extension activity in real time. Using this innovative approach we uncovered several surprising and previously unattainable results. Our preliminary studies show that a single 8- oxoguanine lesion increases the overhang dynamics and accelerates loading of telomeric protein POT1. Consistently, our biochemical studies reveal that an 8-oxoguanine induces robust telomerase activity and processivity on overhangs that are otherwise inaccessible. Aim 1 will define how oxidative lesions impact the telomeric overhang structural diversity, dynamics and accessibility. We will test overhangs with 8-oxoguanine at various positions, along with the most common oxidized thymine lesion, thymine glycol, which imposes a strong block to replication. Aim 2 will examine how oxidative lesions modulate telomerase accessibility and activity. Telomerase activity on overhangs with DNA lesions will be tested in the absence and presence of telomerase processivity factor POT1-TPP1. For aims 1 and 2, complementary biochemical experiments will be performed to validate and interpret the single molecule results. Aim 3 will examine how oxidative lesions modulate telomere length and telomerase recruitment to telomeres in human cells. General oxidative stress will be induced with pro-oxidant conditions, whereas 8-oxoguanine induction at telomeres will be achieved by an innovative fluorogen activated peptide targeting system. Fluorescent in situ hybridization will be used to measure telomere length and to localize telomerase in cells. We will examine telomere length and telomerase recruitment in cells lacking distinct glycosylases that are required to repair specific DNA lesions. These studies will provide crucial insights into how oxidative DNA damage impacts the processing of chromosome end structures. This project will fill a significant void in our understanding of how general oxidative stress and 8- oxoguanine, in particular, alters telomere maintenance. Ultimately, this knowledge will be highly valuable for developing new strategies that 1) preserve telomeres to mitigate the effects of oxidative stress on healthy cells or conversely, that 2) inhibit telomerase in malignant cells to halt proliferation.